Expanding the molecular toolbox for Lactococcus lactis: construction of an inducible thioredoxin gene fusion expression system
© Douillard et al; licensee BioMed Central Ltd. 2011
Received: 6 June 2011
Accepted: 9 August 2011
Published: 9 August 2011
The development of the Nisin Inducible Controlled Expression (NICE) system in the food-grade bacterium Lactococcus lactis subsp. cremoris represents a cornerstone in the use of Gram-positive bacterial expression systems for biotechnological purposes. However, proteins that are subjected to such over-expression in L. lactis may suffer from improper folding, inclusion body formation and/or protein degradation, thereby significantly reducing the yield of soluble target protein. Although such drawbacks are not specific to L. lactis, no molecular tools have been developed to prevent or circumvent these recurrent problems of protein expression in L. lactis.
Mimicking thioredoxin gene fusion systems available for E. coli, two nisin-inducible expression vectors were constructed to over-produce various proteins in L. lactis as thioredoxin fusion proteins. In this study, we demonstrate that our novel L. lactis fusion partner expression vectors allow high-level expression of soluble heterologous proteins Tuc2009 ORF40, Bbr_0140 and Tuc2009 BppU/BppL that were previously insoluble or not expressed using existing L. lactis expression vectors. Over-expressed proteins were subsequently purified by Ni-TED affinity chromatography. Intact heterologous proteins were detected by immunoblotting analyses. We also show that the thioredoxin moiety of the purified fusion protein was specifically and efficiently cleaved off by enterokinase treatment.
This study is the first description of a thioredoxin gene fusion expression system, purposely developed to circumvent problems associated with protein over-expression in L. lactis. It was shown to prevent protein insolubility and degradation, allowing sufficient production of soluble proteins for further structural and functional characterization.
The food-grade bacterium L. lactis subsp. cremoris in conjunction with the Nisin Inducible Controlled Expression (NICE) system [1–3] has been extensively used over the last few decades as a valuable bacterial expression system for large-scale production of homologous or heterologous proteins , metabolic studies , or membrane proteins . The NICE system is based on the well characterized nisin-dependent, quorum-sensing mechanism of L. lactis [2, 3, 7]. It was initially exploited in L. lactis for heterologous protein overexpression and subsequently implemented in several other Gram-positive bacteria [2, 3, 7–10]. Typically, the genetically-engineered strain L. lactis subsp. cremoris NZ9000 is employed as expression host, as its chromosome contains the signal transduction genes nisR and nisK involved in the nisin-induced transcriptional control of the PnisA promoter . Any genes cloned downstream this nisin-inducible promoter PnisA can be expressed in a controlled manner upon addition of nisin to the bacterial culture . However, production of recombinant proteins can be problematic in L. lactis, as over-expressed proteins may be subject to poor expression, stability and/or solubility. Such drawbacks are intrinsically associated with the prokaryotic cell machinery limitations and therefore are inherent to all bacterial expression systems, representing a significant bottleneck in high level production of soluble proteins.
In E. coli, a 'microbial cell factory' of choice for producing heterologous proteins [11, 12], the development of the gene fusion technology proved to circumvent such recurrent and fundamental protein expression problems . This technology involves the linkage of the protein of interest with a carrier protein to generate a fusion protein. Addressing solutions to problematic protein expressions, many fusion expression systems have been engineered and successfully employed, using solubility-enhancing fusion partners such as Schistosoma japonicum glutathione-S-transferase (GST) , E. coli maltose binding proteins (MBP) , Staphylococcus protein A , E. coli N-utilization substance (NusA)  and E. coli thioredoxin (TrxA) [18, 19]. Along with the increasing number of fusion partners used, additional features have been successfully implemented to this technology, thus facilitating protein tagging, purification techniques and tag-mediated proteolytic cleavage [13, 20, 21]. The gene fusion technology provides a substantial palette of applications through the constant expansion of fusion gene expression systems available in E. coli. Nevertheless, the adaptation of these existing fusion partner systems to other expression hosts is sparse, even though significant progress has been made to develop new molecular tools and methods in alternative prokaryotic and eukaryotic expression systems [1, 22, 23]. The expression host L. lactis is currently lacking such a solubility-enhancing expression system to improve its spectrum of biotechnological applications, as L. lactis featured a number of benefits over other expression bacterial hosts, e.g. being a food-grade expression host, and the absence of endotoxins, extracellular proteinases and spores.
As part of our study on the structure-function analysis of lactococcal phage-host recognition and penetration, we attempted to over-express a number of proteins encoded by the lactococcal phage Tuc2009 in L. lactis. However, initial expression studies of individual protein subunits of Tuc2009 phage revealed such proteins often suffer from degradation, poor expression or result in insoluble protein aggregates, also called inclusion bodies (data not shown). The development of a fusion-based gene expression system in L. lactis could provide a novel strategy to express soluble proteins and avoid the use of laborious and spurious renaturation procedures. Among the numerous fusion partners employed, LaVallie et al. described the construction of an E. coli thioredoxin (TrxA) gene fusion system . In most cases, E. coli thioredoxin fusion proteins were soluble, correctly folded and biologically active . The E. coli thioredoxin thus appears to represent a good candidate for an L. lactis fusion-based gene expression system: small size of the fusion partner (11.67 kDa), ability to accumulate in a soluble form at high levels in the cytoplasm, steric accessibility of N- and C-termini of TrxA for protein fusions  and efficient generic protein purification methods available, i.e. immunoprecipitation or affinity chromatography [13, 24].
In the present study, we report on the construction of two new L. lactis thioredoxin-fusion gene expression vectors harbouring the nisin-controlled expression (NICE) system. We evaluated the efficiency of the newly-constructed fusion gene expression system, by producing individual proteins or protein complexes that initially could not be expressed or were not soluble in L. lactis. Our data indicate that the L. lactis thioredoxin-fusion vectors represent a very valuable addition to the L. lactis genetic toolbox, in particular for the over-production of soluble proteins.
Bacterial strains, media, growth conditions and nisin preparation
Bacterial strains and plasmids used in this study
Strains or plasmids
Reference or source
MG1363 containing nisRK genes,
expression host of the NICE system
Nisin producing L. lactis strain
Standard L. lactis expression vector, Cmr
pNZ8048 derivative harbouring the TrxA system, contains a His-tag cloned in frame
pNZ8048 derivative harbouring the TrxA system
pNZ8048 encoding Tuc2009 bppU, bppA, bppL as an operon
pTX8048 encoding Tuc2009 bppU, bppA, bppL as an operon
pNZ8048 encoding Tuc2009 orf40
pTX8048 encoding Tuc2009 orf40
pNZ8048 encoding N-terminal His-tagged Bbr_0140
pNZ8048 encoding C-terminal His-tagged Bbr_0140
pTX8048 encoding Bbr_0140
DNA amplification and cloning
Oligonucleotide primers used in this study
Forward primer of trxA
AGCCTGCAGGATCC CTTGTCGTCGTCGTC ACCAGAAGAATGATGATGATGATGGTG CATATGGCCAGAAC
Reverse primer of trxA flanked by an enterokinase cleavage site and a His-tag
Reverse primer of trxA
Forward primer of bppU for pNZ8048
AGCAGCACTAGT TTAGTGATGGTGATGGTGATG ATTCCGATAAAGTTTTACAATC
Reverse primer of bppL for pNZ8048
Forward primer of bppU for pTX8048
Reverse primer of bppL for pTX8048
Forward primer of orf40 for pNZ8048
AGCAGCACTAGT TTAGTGATGGTGATGGTGATG TAAGTGATAGCCATAAGCAA
Reverse primer of orf40 for pNZ8048
Forward primer of orf40 for pTX8048
Reverse primer of orf40 for pTX8048
TGCATCCCATGG ATCATCACCATCACCATCACCATCAC CATCACAGCCGGATTCTCAAGGAC
Forward primer of bbr_0140 for pNZ8048
Reverse primer of orf40 for pNZ8048
Forward primer of bbr_0140 for pNZ8048
TGCCGTTCTAGA TTAGTGATGGTGATGGTGATGGTG ATGGTGATGTGCGATGTAGCTTTCGATGTGTAG
Reverse primer of bbr_0140 for pNZ8048
Forward primer of bbr_0140 for pTX8048
Reverse primer of bbr_0140 for pTX8048
Construction of thioredoxin gene fusion expression vectors
Construction of expression vectors encoding thioredoxin fusion proteins
The gene encoding the Tuc2009 phage protein Tuc2009 ORF40  was amplified from Tuc2009 DNA and flanked with a C-terminal hexa-histidine tag using primers orf40-F and orf40-R. The PCR product was digested with NcoI and SpeI and then ligated to pNZ8048 cut with NcoI and SpeI. The ligation product was transformed by electroporation into NZ9000 and screened by colony PCR, prior to restriction and sequencing analyses. Similarly, Tuc2009 orf40 was amplified using orf40X-F and orf40X-R, and cloned into the BamHI and SpeI sites of pTX8048. The restriction site BamHI in pTX8048 but also pTX8049 allows the in-frame cloning of a gene of interest, as shown in Figure 1. The three genes encoding the components of the Tuc2009 phage baseplate, i.e. bbpU, bppA and bppL, were amplified from Tuc2009 DNA and flanked with a C-terminal hexa-histidine tag using primers UAL-F and UAL-R. The PCR product was digested with NcoI and SpeI, and cloned into pNZ8048 cut with NcoI and SpeI. Similarly, the DNA region encompassing bppU, bppA and bppL was amplified using UALX-F and UALX-R, and then cloned into the BamHI and SpeI sites of pTX8048. The gene encoding the Bbr_0140 gene product was amplified from Bifidobacterium breve UCC2003 genomic DNA  and flanked with either a C- or an N-terminal hexa-histidine-encoding tag using, respectively, primer combinations 0140C-F and 0140C-R, or 0140N-F and 0140N-R. The PCR product was digested with NcoI and XbaI, and cloned into pNZ8048 cut with NcoI and XbaI. Similarly, Bbr_0140 was amplified using 0140X-F and 0140X-R, and subsequently cloned into BamHI and XbaI-restricted pTX8048.
Protein expression assay
L. lactis NZ9000 cells harbouring one of the various plasmid constructs described in the Methods section were propagated overnight at 30°C in M17 broth  containing 0.5% (w/v) D-glucose and supplemented with 5 μg/ml chloramphenicol. Fresh GM17 media supplemented with 5 μg/ml chloramphenicol was inoculated with a 1/50 (v/v) overnight liquid culture and incubated at 30°C. When the optical density at 600 nm reached 0.4, protein expression was induced by the addition of nisin to a final concentration of 0.2% (v/v) . Liquid culture was further incubated at 30°C for 4 hours and bacterial cells were harvested by centrifugation (3000 × g for 20 min at 4°C). Bacterial cell pellets were washed in 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0 and stored at -80°C until further use .
Fractionation, SDS-PAGE, immunoblotting analysis and protein assays
Protein samples were prepared as described by Bahey-El-Din et al. . Bacterial pellets were resuspended in 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0 supplemented with 30 mg/ml lysozyme and incubated for 30 min on ice. Cell preparations were then sonicated (8 × 10 sec with 10 sec on ice between each cycle) at maximum amplitude (MSE Soniprep 150, Sanyo). Insoluble and soluble fractions were separated by centrifugation at 14, 000 × g for 10 min at 4°C and stored at -20°C for further analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as previously described . Proteins from 12.5% acrylamide gels were then transferred onto a PVD membrane (Millipore, UK) by electroblotting . Mouse polyclonal antibodies directed against the poly-histidine tag or rabbit polyclonal antibodies directed against BppU, BppA and BppL were used as primary antibody . Monoclonal anti-mouse or anti-rabbit antibodies coupled to horseradish-peroxidase (Sigma, USA) were used as secondary antibody. The membrane was developed using hydrogen peroxide and 4-chloro-1-naphthol (Sigma). His-tagged proteins were purified using the PrepEase® Histidine-tagged Protein Purification kit (USB, OH, USA). Protein content was measured using the Bio-Rad Protein Assay (Germany), based on the Bradford protein quantification method.
Enzymatic cleavage of TrxA fusion proteins using enterokinase
The fusion protein TrxA-Bbr_0140 was expressed and His-tagged purified as described above. Purified enterokinase from calf intestine (Roche GmbH, Germany) was used to cleave TrxA-Bbr_0140 according to manufacturer's instructions. TrxA-Bbr_0140 was dialyzed against 50 mM Tris buffer, pH 8.0, since phosphate buffer is known to significantly reduce enterokinase activity as indicated in the user's manual. Typically, 25 μg TrxA-Bbr_0140 was incubated with 0.6 μg enterokinase for 16 h at 20°C or 37°C (enterokinase: TrxA-Bbr_0140 ratio = 1:42). We tested the effect of SDS on enterokinase cleavage efficiency as recommended in the manufacturer's manual, by supplementing the reaction mixture with 0.1% (w/v) SDS. An equal volume of 2X SDS-PAGE loading buffer was added and the samples were boiled at 95°C for 5 min to inactivate enterokinase. The cleaved protein products were analyzed by SDS-PAGE.
Results and discussion
Description of the two L. lactis thioredoxin-fusion expression vectors
The two L. lactis thioredoxin-fusion expression vectors, called pTX8048 and pTX8049 (Figure 1) were employed in an attempt to express a number of proteins encoded by the lactococcal phage Tuc2009 and B. breve UCC2003. The anticipated translational fusions in both plasmids are placed under the transcriptional control of the nisin-inducible promoter PnisA, ensuring a tight control of protein expression in L. lactis . The original ribosome binding site present in plasmid pNZ8048 was retained to ensure efficient translation of the fusion protein (Figure 1) as it had previously been reported that low expression of proteins may be due to inefficient translational initiation of mRNA . The E. coli thioredoxin trxA represents the N-terminal portion of the fusion protein (Figure 1), promoting an efficient initiation of translation as previously described . In addition, plasmid pTX8048 has been designed to join the thioredoxin C-terminus to the recombinant protein N-terminus with an amino-acid linker (SSGDDDDKGS) adapted from LaVallie et al. , consisting of serine (S), glycine (G), aspartic acid (D) and lysine (K) residues and a highly-specific enterokinase cleavage site (DDDDK) previously used in an E. coli thioredoxin fusion system  (Figure 1). The (SSG)X5(GS) residues act as flexible joints within the fusion protein connecting the thioredoxin C-terminus to the recombinant protein N-terminus. It facilitates access to the enterokinase cleavage site (DDDDK), to facilitate release of the mature protein . In pTX8048, the thioredoxin-specifying sequence was modified to include a C-terminal hexa-histidine encoding tag, also termed 'Histidine-patch thioredoxin' in the originally developed E. coli fusion expression systems [36, 37] enabling the purification of the fusion protein by Ni-TED affinity chromatography. The thioredoxin and the hexa histidine-tag are located upstream of the enterokinase cleavage site and can therefore be removed from the protein of interest (Figure 1). In comparison, pTX8049 only contains the E. coli thioredoxin gene trxA followed by a multiple cloning site to insert a gene of interest in an in-frame manner (Figure 1). The lack of additional purification tags or linkers in pTX8049 allows a greater level of flexibility in designing and constructing original fusion proteins, i.e. addition, choice and location of purification tags , peptide linkers  and specific cleavage sites (tobacco etch virus protease cleavage site, thrombin or factor Xa)  (Figure 1). Methods and performances to over-produce proteins using pTX8049 are identical to pTX8048, as they both share the same pNZ8048 backbone and range of bacterial expression hosts.
Production of Tuc2009 ORF40 as a fusion protein
Production and purification of the Tuc2009 phage baseplate in L. lactis NZ9000
Over production of Bifidobacterium breve UCC2003 Bbr_0140 using pTX8048
Enterokinase cleavage of the thioredoxin fusion protein TrxA-Bbr_0140
The thioredoxin gene fusion system represents an attractive system to over-produce and purify proteins in L. lactis that exhibit poor or no expression, or produce insoluble proteins using conventional expression vectors. In this study, we have described the construction of an L. lactis Trx-fusion expression system and demonstrated its applicability by over-producing and purifying various proteins or complexes as soluble thioredoxin fusions. The benefits of the original E. coli thioredoxin fusion expression system have previously been demonstrated [18, 19], and in this report we have shown that these are also applicable to the expression host L. lactis, when combined with the NICE system. This expression and purification tool offers a wide spectrum of applications in L. lactis and also other Gram-positive bacteria that can accommodate the NICE system, such as L. plantarum . Although our study does not show the functionality of the overexpressed proteins, we are confident that the majority of such proteins are biologically active as based on numerous peer-reviewed studies using the original NICE system, as reviewed by Mierau et al. . The protein production levels obtained in L. lactis using the thioredoxin fusion gene expression system allow further structural and biochemical analysis, such as X-ray crystallography analysis, antibody production, protein-protein interaction assays, and enzymatic assays.
The study described in this manuscript is supported by D. van Sinderen's Science Foundation Ireland Principal Investigator grant (Ref. No. 08/IN.1/B1909).
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